JPS5948080A - Immobilized composite enzyme - Google Patents

Abstract

PURPOSE:To obtain an immobilized composite enzyme having excellent properties such as small diffusion resistance of the substrate, by depositing and fixing plural enzymes in layers on the surface of a carrier. CONSTITUTION:The objective immobilized composite enzyme contains plural enzymes fixed integratedly in layers to the surface of a carrier. The immobilized enzyme can be prepared e.g. by fixing an enzyme to a carrier through crosslinks or covalent bonds using a reagent such as a crosslinking agent, and fixing the second enzyme to the immobilized first enzyme through ionic bond to cover the first enzyme layer with the second enzyme layer. The carrier is, e.g. platinum coated with aminated polyvinyl alcohol, and the composite enzyme is, e.g. a combination of glucose isomerase and invertase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an immobilized complex enzyme used as a catalyst or the like.

In recent years, enzymes have been increasingly used in various fields such as the pharmaceutical industry and the food industry. Reactions catalyzed by enzymes proceed under mild conditions of room temperature and pressure, and are less likely to cause pollution than conventional chemical reactions. Furthermore, enzymes also have the advantage of not causing side reactions due to their excellent substrate specificity, which greatly reduces the processing of reaction products. However, enzymes are water-soluble, and conventionally the enzyme reaction was carried out with the enzyme dissolved in water, so after the reaction was completed, only the enzyme was separated and recovered from the reaction solution and the enzyme was reused. This was technically extremely difficult. Since enzymes are expensive, the inability to reuse enzymes is a significant cost disadvantage. Therefore, in order to eliminate such drawbacks, it has been proposed to immobilize the enzyme, such as by modifying the enzyme in some way to make it water-insoluble.

Enzyme immobilization methods proposed so far can generally be divided into three methods: carrier binding methods, crosslinking methods, and entrapment methods. In the most common cases, the carrier binding method is a method in which the enzyme is bound to a carrier to make it water-insoluble, and the crosslinking method is a method in which the enzyme is reacted with a reagent (crosslinking agent) having two or more functional groups, and the enzyme is A method of binding enzymes with a cross-linking agent to make them water-insoluble, and an entrapment method involve enclosing the enzyme in a fine gel lattice (lattice type) or covering it with a semipermeable polymer film (microcapsules). type) method.

If immobilized enzymes obtained by these methods are used, enzymatic reactions can be carried out in a continuous manner, whereas conventional enzyme reactions could only be carried out in a batch manner (hatch type). Furthermore, since the enzyme can be used for many reactions until it is deactivated or denatured, it is very advantageous in terms of cost. Furthermore, by utilizing the substrate specificity of enzymes, it will be possible to create sensors that detect specific substances.

By the way, a group of enzymes used in a series of enzymatic reactions of two or more steps is called complex enzymes, but immobilized enzymes in which multiple enzymes are immobilized in a complex manner, that is, immobilized complex enzymes, have recently been used. It has become. When immobilized complex enzymes are used for enzymatic reactions, one type of enzyme can perform not only one-step enzymatic reaction but also two or more steps of enzymatic reactions. This method is very convenient when using two or more types of immobilized enzymes.

Conventionally, the immobilization of a complex enzyme has been carried out by enclosing the complex enzyme in a polymer matrix at once.

However, the immobilized complex enzyme obtained by this method has a heterogeneous reaction system due to factors such as only specific different enzymes coming close to each other or specific enzymes being localized in the polymer matrix. This often results in a reaction system with large diffusion resistance, which has caused various problems such as slowing down the reaction rate of the enzyme reaction.

Furthermore, since the optimal immobilization conditions for each enzyme in a complex enzyme are often different, if all the enzymes are wrapped in a polymer matrix at once, some of the enzymes in the complex enzyme may be deactivated or denatured, resulting in a series of In many cases, the enzymatic reaction did not proceed, that is, the enzyme did not show any activity as a complex enzyme.

The present invention was made in view of the above circumstances, and aims to provide an immobilized complex enzyme with excellent performance such as low substrate diffusion resistance.

That is, the present invention provides an immobilized complex enzyme in which a plurality of enzymes are immobilized in a complex manner, and is characterized in that the plurality of enzymes are all fixed in a layered manner on the surface of a carrier in the form of a membrane. The gist is immobilized complex enzymes. The present invention will be explained in detail below.

In the immobilized composite enzyme according to the present invention, a plurality of enzymes (complex enzymes) are sequentially stacked and fixed in a membrane shape, so that all other enzyme membranes are sandwiched between the carrier and the outermost enzyme membrane. ,
Only specific dissimilar enzymes are close to each other, and specific enzymes are not localized.

Therefore, when the immobilized complex enzyme according to the present invention is used in an enzymatic reaction, the reaction rate is high. Moreover, when this is used in a sensor, the response speed becomes faster. When producing an immobilized complex enzyme, the enzymes are sequentially immobilized in a membrane shape, so each enzyme can be immobilized using optimal immobilization conditions. Therefore, there is less possibility that the enzyme will be deactivated or denatured, and it will almost never occur that the produced immobilized composite enzyme does not exhibit activity as a composite enzyme.

The immobilized enzyme according to the present invention is produced, for example, as follows. After one of the enzymes in the complex is bound ionically and physically, the enzyme and the carrier are immobilized by crosslinking or covalent bonding using a reagent such as a crosslinking agent on the surface of the carrier to form an enzyme membrane. Next, the immobilized enzyme and other enzymes are ionically bonded to form a rough enzyme membrane on the outside of the enzyme membrane. In this way, an immobilized composite enzyme having two layers of enzyme membranes can be obtained. If enzyme membranes are sequentially formed in the same manner, an immobilized composite enzyme having three or more layers of enzyme membranes can be obtained. As carrier, sieve-berliLe
Platinum coated with IRA-94 or aminated polyvinyl alcohol is used, and as a composite enzyme, for example,
Introduction of glucose isomerase and inherutase,
Alternatively, a combination of glucose oxidase and invertase is used. When using a crosslinking agent, glucuraldehyde or the like is used. When ionically bonding one of the complex enzymes, it is preferable to perform the following procedure, focusing on the isoelectric point of the enzyme, for example. When a carrier and this enzyme are mixed in a solution with a pI different from the isoelectric point pI of an enzyme, an ionic bond is formed between the two due to the difference in hydrogen index (ionic index) between pH and pI. It can be done in the same way when another enzyme is ionically bound to the previously immobilized enzyme.In other words, it can be done at a pH different from the isoelectric point p2 of the other enzyme.
An enzyme solution is prepared by dissolving the enzyme in a solution of When the carrier on which the first enzyme membrane is formed is immersed in this enzyme solution, ionic bonds are formed between the previously immobilized enzyme and other enzymes.
．． A second enzyme membrane is formed outside the first enzyme membrane. The third and subsequent enzyme membranes can be formed in the same manner. After ionically bonding enzymes of different types, they may be fixed by crosslinking or covalent bonding using a reagent such as a crosslinking agent. This reduces the possibility that the enzyme will be desorbed.

The immobilized complex enzyme according to the present invention is constructed as described above, in which a certain enzyme among a plurality of enzymes is immobilized on the surface of a carrier in the form of a film, and other enzymes are sequentially placed on top of it in the form of a film. Since the dirt is laminated and fixed, the reaction speed becomes faster when the dirt is used in an enzyme reaction, and the response speed becomes faster when used in a sensor. In addition, it almost never happens that the produced immobilized complex enzyme does not exhibit activity as a complex enzyme.

Next, Examples and Comparative Examples will be described.

[Example 1] An immobilized composite enzyme having a 2N enzyme film formed on the surface of a carrier was prepared in the following manner. However, a combination of glucose isomerase and inherutase was used as the complex enzyme, and static berlite I R was used as the carrier.
A94 was used.

Glucose isomerase 30μ pH5,0,0,O
IM's acetic acid f! Enzyme/8/& was prepared by dissolving it in 20ml overnight. Amberlite l RA
-94 was thoroughly washed and dried, and 5 g of the obtained dried product was branched into the previously prepared enzyme/g liquid at 5°C. Next, while maintaining the enzyme solution at 5°C,
A first immobilized preparation in which glucose isomerase was ionically immobilized on Ambe-rlite IRA 94 was obtained by performing rotational stirring for a period of time. After removing unimmobilized residual enzyme by filtration, the first immobilized preparation was thoroughly washed with OIM acetate buffer, pH 5.0.0. After this, the first immobilized specimen is immersed in a cold 1% glutaraldehyde aqueous solution for 30 minutes, and then air-dried at 5°C, so that the glucose isomerase and the carrier are ionically bonded. A second immobilized specimen was obtained which was cross-linked with the same. Next, add inherutase 35■ to pH 5.5,
An enzyme solution was prepared by dissolving the enzyme in 20n+1 of O, OIM acetate buffer. The second immobilized specimen was immersed in this enzyme/g solution, and inherutase was immobilized at 5°C for 12 hours, resulting in a third immobilization in which inherutase was ionically bound to glucose isomerase. I got a specimen. This third immobilized preparation was added to a cold 1% aqueous glutaraldehyde solution for 30 min.
After soaking for a minute, it was air-dried to obtain an immobilized complex enzyme.

(9) An immobilized composite enzyme of Comparative Example 1 was prepared by enclosing the same amount of glucose isomerase and invertase as contained in the immobilized composite enzyme of Example 1 in a polyacrylamide gel.

The immobilized complex enzymes of Example 1 and Comparative Example 1 were added to 1σ3M sucrose-pH5,5,0,01M #acid buffer, an enzyme reaction was performed, and changes in enzyme activity (reaction activity) over time were measured. . However, the enzyme activity was expressed as a specific activity, where the activity of the same amount of enzyme immobilized on the immobilized complex enzyme was taken as 100. The measurement results are shown in Table 1.

From Table 1, it can be seen that the immobilized complex enzyme of Example 1 has a higher enzyme activity (10) than that of Comparative Example 1 and is stable.

[Example 2] An immobilized composite enzyme having two layers of enzyme membranes formed on the surface of a carrier was prepared in the following manner. However, a combination of glucose oxidase and inherutase was used as the composite enzyme, and a platinum plate coated with aminated polyvinyl alcohol was used as the carrier.

Glucose oxidase 40■ pH 5,0,0,0
1M acetic acid rim fi liquid 2Qmj! An enzyme solution was prepared by dissolving the enzyme in A platinum plate (5 m long, 5 inches wide, 70 μm thick) coated with aminated polyvinyl alcohol was placed in this enzyme solution.
m) and rotary stirring was performed for 10 hours while maintaining the temperature at 5°C. 5 μp of a 2% albumin aqueous solution and 1 µp of a 1% glutaraldehyde aqueous solution were placed on the surface of the platinum plate treated as described above.
0 μl of each was applied and then air-dried. 50 mg of invertase was dissolved in 20 ml of acetate buffer at pH 5,2,0,01M.

An air-dried platinum plate was immersed in this inherutase solution, and
Rotary stirring was carried out for 12 hours while the temperature was maintained at .degree.

(11) The obtained enzyme immobilized product (immobilized sample) was immersed again in a 1% cold glutaraldehyde solution for about 20 minutes, and then
The immobilized complex enzyme was obtained by air-drying it while keeping it at ℃.

The obtained immobilized complex enzyme was used as a sensor and a sucrose solution (
The concentration of the reaction solution) was determined. Comparative Example 2 is a combination of an immobilized enzyme in which glucose oxidase is immobilized on a platinum plate using glutaraldehyde and invertase that is not immobilized, and this is used as a sensor, and invertase is dissolved in 1 g WM of sucrose. The concentration of the sucrose solution was determined. The sucrose concentration was 10-
Table 2 shows the response speed (seconds) and output current value (μA) at 3M, 10M, and 10M.

(Margin below) (12) Table 2 From Table 2, when Example 2 is used, the response speed is faster at all concentrations and the output current value is higher than when Comparative Example 2 is used. I understand that. Therefore, when the immobilized complex enzyme of Example 2 is used in a sensor, a sensor with high sensitivity can be obtained.

Agent: Patent Attorney Takehiko Matsumoto (13)

Claims (3)

[Claims] (1) An immobilized composite enzyme in which a plurality of enzymes are immobilized in a complex manner, wherein all of the plurality of enzymes are layered and immobilized on the surface of a carrier in the form of a membrane. enzyme. (2) The immobilized complex enzyme according to claim 1, wherein the enzyme is immobilized in the form of a membrane through ionic bonding. (3) The immobilized complex enzyme according to claim 1, wherein the enzyme is immobilized in the form of a membrane through a combination of ionic bonding and cross-linking.